Multiple drug resistance (MDR) describes a broad cross-resistance of tumor cells to a variety of "natural product" compounds of widely differing structure and mechanism of action. One type of MDR (Pgp). Certain membrane-active drugs and biologicals modulate MDR, and appear to be more toxic to the Pgp-MDR cells than to their drug-sensitive counterparts. Another type of MDR is "atypical" (at-MDR) in that the cells are unimpaired in their transport of drugs, not affected by modulators, and do not overexpress the pgp gene or Pgp. The essential nuclear enzyme, topoisomerase II, appears to be altered in at-MDR. The long-term goal of this research is to understand the basis of MDR and its modulation in tumor cells. How does Pgp mediate MDR? Does it bind drug and extrude it or does it alter membrane fluidity, endocytosis or exocytosis (i.e., membrane traffic)? How can modulators both enhance the toxic activity of drugs in MDR cells and yet be more toxic to such cells than to their drug-sensitive counterparts? What is the mechanism of at-MDR? To answer these questions, six specific aims are proposed. The first is to measure membrane traffic in established Pgp-MDR cell lines, while the second is to compare the cellular pharmacology and membrane traffic properties of cells transfected with normal pgp cDNAs and those mutagenized at nucleotide and putative drug-binding sites. A third aim is to determine by structure-activity studies whether modulators of Pgp- MDR (reserpine and verapamil) work by binding to Pgp or by altering membrane traffic. The fourth aim is to determine the basis for the increased sensitivity of Pgp-MDR cells to verapamil, tumor necrosis factor (TNF), and the alkalinizing effects of ouabain by studies of membrane traffic in these cells and by development of cell lines resistant to these agents. A fifth aim is to determine the mechanism of at-MDR by measuring drug binding to topoisomerase II, drug-induced DNA cleavage patterns mediated by topoisomerase II, and protein kinase C activity. The last aim is to determine the relationship between topoisomerase II and TNF in the potentiation of topoisomerase II-active drugs by TNF. It is anticipated that the studies outlined in this proposal will provide insights into the design of new drugs and therapeutic modalities to overcome these forms of resistance.